The objective of this proposal is to test the hypothesis that the reaction of myoglobin with hydrogen peroxide or other biological oxidants leading to the formation of the high oxidation state of this hemoprotein, ferrylmyoglobin, is an early functional change with toxicological implications for muscle tissue. We propose that the chemical reactivity with which ferrylmyoglobin is endowed is a basis for ensuing tissue oxidative damage. This hypothesis appears tenable since [I] ferrylmyoglobin occurs in cells and perfused heart, [II] myoglobin is oxidized by physiological peroxides in myocytes, and [III] ferrylmyoglobin is reduced efficiently in vitro by several antioxidants. This proposal is to study chemical mechanisms inherent in the formation of the high oxidation state of myoglobin, its recovery to a functional hemoprotein, and its implications for myocardial toxicity from studies with myocytes and mitochondria. Specific Aim 1 is to provide an understanding of the redox transitions encompassed by the two-electron oxidation of myoglobin to ferrylmyoglobin as well as to characterize the individual chemical reactivities of the electrophilic centers in ferrylmyoglobin. Specific Aim 2 is concerned with the recovery of ferrylmyoglobin to a functional hemoprotein by different electron donors by one-electron transfer processes as well as the kinetic and environmental factors that govern these reactions. The studies devised in Specific Aim 3 are directed at evaluating the role of mitochondrially generated hydrogen peroxide on myoglobin oxidation and the implications of these reactions for muscle oxidative injury. [l] The mechanistic and catalytic aspects implied in myoglobin oxidation will be evaluated by experimental models involving one- and two-electron oxidation of the hemoprotein. [2] The electron-transfer reactions leading to myoglobin recovery will be assessed with a range of electron donors with different physico-chemical properties. [3] The biological implications of the reaction between myoglobin and hydrogen peroxide will be determined with isolated heart mitochondria and myocytes and assessed in terms of the reactivity of ferrylmyoglobin towards membrane antioxidants and cytoskeletal proteins. Absorption spectroscopy will be used to monitor the different oxidation states of myoglobin and oxidative reactions involving protein-heme and drug-heme covalent bindings. ESR with the spin trapping technique will be used to detect oxygen-derived radicals and thiyl radicals. Direct ESR with will be used to identify free radicals derived from electron donors, such as ascorbate, vitamin E, and ubiquinol. Direct ESR assisted by flow experiments will be used to identify protein- derived radicals. HPLC methods with different detection modes will be used as follows: [a] UV detection: determination of glutathione and oxidation products of vitamin E, ubiquinol, and water-soluble electron products. [b] Electrochemical detection: identification and red ox properties of oxidation products of different electron donors. [c] Fluorometric detection: determination of vitamin E. SDS-PAGE analysis and Western blot analysis will be used to study cross-linking of cytoskeletal proteins with myoglobin.